Table 1.
Scoring system for the treatment with GA on the CCl4-induced hepatotoxicity.
Table 2.
Scoring system for the treatment with GA on the CCl4-induced liver fibrosis.
Figure 1.
Effects of GA on histopathological changes by CCl4 in mice were evaluated in sections stained with hematoxyline-eosin.
Mice in all groups were treated as the same with the front method. The animals were sacrificed 24 h after the last CCl4 administration and the liver was removed, fixed and embedded in paraffin. Sections were stained with hematoxyline-eosin (H-E, 200×). (A) Liver tissue of a control mouse. (B) Liver tissue of a mouse treated with CCl4, presenting severe hepatocyte necrosis with neutrophil clusters and mononuclear cells infiltration (arrow 2) around the portal vein. (C) Liver tissue of a mouse treated with silymarin (100 mg/kg, i.g.), showing mild hepatocyte necrosis with inflammatory cell infiltration and steatosis (arrow 1) around the portal vein. (D) Liver tissue of a mouse treated with GA (25 mg/kg, i.g.), showing moderate hepatocyte necrosis with inflammatory cell infiltration and moderate steatosis (arrow 1). (E) Liver tissue of a mouse treated with GA (50 mg/kg, i.g.), showing mild steatosis (arrow 1) around centrilobular and midzone region. (F) Liver tissue of a mouse treated with GA (100 mg/kg, i.g.), showing mild hepatocyte necrosis with inflammatory cell infiltration (arrow 2) and severe steatosis (arrow 1).
Figure 2.
The inflammation score was evaluated in the livers of surviving animals by certified pathologist in a blinded fashion.
Cont, normal control; CCl4, CCl4 alone; Sil, CCl4+100 mg/kg silymarin; GA25, CCl4+25 mg/kg GA; GA50, CCl4+50 mg/kg GA; GA100, CCl4+100 mg/kg GA. Datas are presented as the mean ± SD (n = 8) in each group. *** Significantly different from the control at p<0.001; ## Significantly different from the CCl4 at p<0.01; ### Significantly different from the CCl4at p<0.001.
Table 3.
Effects of the treatment with GA on the CCl4-induced liver fibrosis and lipid peroxidation.
Figure 3.
Effects of GA on serum and homogenate MAO changes by CCl4 in mice.
Mice in all groups were treated as the same with the front method. Liver fibrosis was determined by quantifying the serum activities of MAO (Fig. 3A) as well as liver homogenate MAO (Fig. 3B). Cont, normal control; CCl4, CCl4 alone; Sil, CCl4+100 mg/kg silymarin; GA25, CCl4+25 mg/kg GA; GA50, CCl4+50 mg/kg GA; GA100, CCl4+100 mg/kg GA. Datas are presented as the mean± SD (n = 8) in each group. *** Significantly different from the control at p<0.001; # Significantly different from the CCl4 at p<0.05; ### Significantly different from the CCl4 at p<0.001.
Figure 4.
Effects of GA on histopathological changes by CCl4 in mice were evaluated in sections stained with collagenous fiber.
Mice in all groups were treated as the same with the front method. The animals were sacrificed 24 h after the last CCl4 administration and the liver was removed, fixed and embedded in paraffin. Sections were stained with collagenous fiber (V-G, 200×). (A) Liver tissue of a control mouse. (B) Liver tissue of a mouse treated with CCl4, numerous fibrocytes appeared at the periphery of the lesions, and the collagen fibers became longer and thicker, presenting severe liver fibrosis and severe hepatocyte necrosis with inflammatory cell infiltration around the portal vein. (C) Liver tissue of a mouse treated with silymarin (100 mg/kg, i.g.), showing broad-develop septa and moderate liver fibrosis around the portal vein. (D) Liver tissue of a mouse treated with GA (25 mg/kg, i.g.), showing slender septa linking hepatic vein and mild hepatic fibrogenesis. (E) Liver tissue of a mouse treated with GA (50 mg/kg, i.g.), showing mild fibrosis around centrilobular and midzone region. (F) Liver tissue of a mouse treated with GA (100 mg/kg, i.g.), showing moderate fibrosis. Arrow 1 shows collagen fibers which was stained red, while arrow 2 shows inflammatory cell infiltration.
Figure 5.
Effects of GA on histopathological changes by CCl4 in mice were evaluated in sections stained with Masson.
Mice in all groups were treated as the same with the front method. The animals were sacrificed 24 h after the last CCl4 administration and the liver was removed, fixed and embedded in paraffin. Sections were stained with Masson (200×). (A) Liver tissue of a control mouse. (B) Liver tissue of a mouse treated with CCl4, presenting severe liver fibrosis (arrow 1) and ballooning degeneration (arrow 2) aroud the portal vein. (C) Liver tissue of a mouse treated with silymarin (100 mg/kg, i.g.), presenting well-developed septa, showing moderate liver fibrosis (arrow 1) and hepatocyte necrosis with inflammatory cell (arrow 2) infiltration around the portal vein. (D) Liver tissue of a mouse treated with GA (25 mg/kg, i.g.), presenting slender septa linking hepatic veins, showing mild liver fibrosis (arrow 1) and severe sreatosis (arrow 2). (E) Liver tissue of a mouse treated with GA (50 mg/kg, i.g.), presenting slender septa, showing mild fibrosis (arrow 1) and sreatosis (arrow 2) around centrilobular and midzone region. (F) Liver tissue of a mouse treated with GA (100 mg/kg, i.g.), showing severe fibrosis (arrow 1) and sreatosis (arrow 2). Arrows show collagen fibers, which were stained blue (Masson trichrome staining).
Figure 6.
Fibrosis score was evaluated in the liver sections stained with V-G of surviving animals by certified pathologist in a blinded fashion. Cont, normal control; CCl4, CCl4 alone; Sil, CCl4+100 mg/kg silymarin; GA25, CCl4+25 mg/kg GA; GA50, CCl4+50 mg/kg GA; GA100, CCl4+100 mg/kg GA. Datas are presented as the mean ± SD (n = 8) in each group. *** Significantly different from the control at p<0.001; # Significantly different from the CCl 4 at p<0.05; ## Significantly different from the CCl4 at p<0.01; ### Significantly different from the CCl4 at p<0.001.
Figure 7.
Fibrosis score was evaluated in the liver sections stained with Masson of surviving animals by certified pathologist in a blinded fashion. Cont, normal control; CCl4, CCl4 alone; Sil, CCl4+100 mg/kg silymarin; GA25, CCl4+25 mg/kg GA; GA50, CCl4+50 mg/kg GA; GA100, CCl4+100 mg/kg GA. Datas are presented as the mean ± SD (n = 8) in each group. *** Significantly different from the control at p<0.001; # Significantly different from the CCl4 at p<0.05; ## Significantly different from the CCl4 at p<0.01.
Table 4.
Antioxidative effects of the treatment with GA on the CCl4-induced liver fibrosis.
Figure 8.
Effects of GA on CCl4 bioactivation related Nrf2 expression in cytoplasm.
Mice in all groups were treated as the same with the front method. The animals were sacrificed 24 h after the last CCl4 administration. (A) The expression of Nrf2 and GAPDH in the liver microsomes was determined by western blotting. GAPDH was used as an internal control. (B) Quantitative analysis of the Nrf2 proteins. Cont, normal control; CCl4, CCl4 alone; Sil, CCl4+100 mg/kg silymarin; GA25, CCl4+25 mg/kg GA; GA50, CCl4+50 mg/kg GA; GA100, CCl4+100 mg/kg GA. Data are presented as the mean± SD for three independent experiments, performed in triplicate. *** Significantly different from the control at p<0.001; ### Significantly different from CCl4 at p<0.001.
Figure 9.
Effects of GA on CCl4 bioactivation related Nrf2 expression in nuclear.
Mice in all groups were treated as the same with the front method. The animals were sacrificed 24 h after the last CCl4 administration. (A) The expression of Nrf2 and GAPDH in the liver microsomes was determined by western blotting. GAPDH was used as an internal control. (B) Quantitative analysis of the Nrf2 proteins. Cont, normal control; CCl4, CCl4 alone; Sil, CCl4+100 mg/kg silymarin; GA25, CCl4+25 mg/kg GA; GA50, CCl4+50 mg/kg GA; GA100, CCl4+100 mg/kg GA. Data are presented as the mean± SD for three independent experiments, performed in triplicate. *** Significantly different from the control at p<0.001; ### Significantly different from CCl4 at p<0.001.
Figure 10.
Effect of GA on the mRNA expression levels of SOD3, CAT, and GPX2 genes.
Total RNA was extracted from mice liver tissues of GA-treated, CCl4-administrated, silymarin-treated, and control group animals after sacrificed. qRT-PCR was performed for analysis of mRNA expression levels of SOD3, CAT, and GPX2. (A) qRT-PCR results for analysis of SOD3 mRNA. (B) qRT-PCR results for analysis of CAT mRNA. (C) qRT-PCR results for analysis of GPX2 mRNA. Cont, normal control; CCl4, CCl4 alone; Sil, CCl4+100 mg/kg silymarin; GA25, CCl4+25 mg/kg GA; GA50, CCl4+50 mg/kg GA; GA100, CCl4+100 mg/kg GA. Data are presented as the mean± SD for three independent experiments, performed in triplicate.*** Significantly different from the control at p<0.001; ### Significantly different from CCl4 at p<0.001.
Figure 11.
Inhibitory effects of GA on FeCl2-ascorbic acid stimulated lipid peroxidation and DPPH radical scavenging activity.
(A) The mouse liver homogenates were stimulated with FeCl2-ascorbic acid in the presence or absence of GA. The lipid peroxidation was measured as described in Section 2. Control value of lipid peroxidation in liver homogenates. (B) The DPPH radical scavenging activity was determined by the DPPH assay in the presence or absence of GA and the scavenging activity was measured as described in Section 2. The value is presented as the mean of the percentage inhibition±SD for three independent experiments, performed in triplicate. *Significantly different from the control (GA = 0 µg/ml) at p<0.05.
Figure 12.
Potential mechanisms for anti-fibrosis mechanism of GA against CCl4-induced liver fibrosis in mice.